Molecular plasma deposition of colloidal materials

ABSTRACT

A molecular plasma discharge deposition method for depositing colloidal suspensions of biomaterials such as amino acids or other carbon based substances onto metal or nonmetal surfaces without loss of biological activity and/or structure is described. The method is based on generating a charged corona plasma which is then introduced into a vacuum chamber to deposit the biomaterial onto a biased substrate. The deposited biomaterials can be selected for a variety of medical uses, including coated implants for in situ release of pharmaceuticals.

This application claims benefit of U.S. provisional application Ser. No.60/777,104 filed Feb. 27, 2006, the entire contents of which are hereinincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to an apparatus and process for using coronadischarge to deposit colloidally suspended molecules onto substratesurfaces. The method is applicable to deposition of organic andinorganic compounds, particularly to proteins and related biologicalcompounds of interest onto selected substrates with little or no loss ofnative structure or activity.

2. Description of Background Art

There is increasing interest in the immobilization of biologicallyactive substances onto various substrates without significant alterationof function or desired activity. Surfaces coated with antibiotics, forexample, are typically prepared by dipping or paint processes, whichoften result in poor adhesion, incomplete surface wetting or pooradhesion.

Ionic plasma deposition (IPD) methods have been extensively developedand used in coating processes, predominately with the objective ofproducing highly adhesive coatings and customized surfacecharacteristics. Attention has recently focused on preparing coatedsurfaces that are biocompatible, such as those suitable for medicalimplants where the coatings enhance cell adhesion or where antimicrobialcoatings are important in avoiding potential sepsis after surgery.

Corona discharge is a well-known phenomenon which has long been observedin nature and traditionally used in a number of commercial andindustrial processes. It is currently used in ozone production, controlof surface generated electrical charges and in photocopying. Electriccorona discharge has also been used to modify surfaces, particularly forplastic articles to improve surface characteristics, as described inU.S. Pat. No. 3,274,089. An electrostatic coating process involving acorona discharge of a liquid or powdered material is described as acoating method, U.S. Pat. No. 4,520,754.

A corona is generated when the potential gradient is large enough at apoint in the fluid to cause ionization of the fluid so that it becomesconductive. If a charged object has a sharp point, the air around thatpoint will be at a much higher gradient than elsewhere. Air near anelectrode can become ionized (partially conductive), while regions moredistant do not. When the air near the point becomes conductive, it hasthe effect of increasing the apparent size of the conductor. Since thenew conductive region is less sharp, the ionization may not extend pastthe local region. Outside the region of ionization and conductivity, thecharged particles slowly find their way to an oppositely charged objectand are neutralized.

Corona discharge usually involves two asymmetric electrodes; one highlycurved, e.g., a needle tip or a small diameter wire, and one of lowcurvature, e.g., a plate, or a ground. The high curvature ensures a highpotential around the electrode, providing for the generation of aplasma. If the geometry and gradient are such that the ionized regioncontinues to grow instead of stopping at a certain radius, a completelyconductive path may be formed, resulting in a momentary spark or acontinuous arc.

Coronas may be positive or negative. This is determined by the polarityof the voltage on the highly-curved electrode. If the curved electrodeis positive with respect to the flat electrode a positive corona exists;otherwise the corona is negative. The physics of positive and negativecoronas are strikingly different. This asymmetry is a result of thegreat difference in mass between electrons and positively charged ions,with only the electron having the ability to undergo a significantdegree of ionizing inelastic collision at ordinary temperatures andpressures.

Corona discharge systems have been used to activate chemical compounds,generally to deposit polymers and polymerizable monomers formed within acorona discharge onto surfaces as protective coatings; as described inU.S. Pat. No. 3,415,683. A corona discharge reactor for chemicallyactivating constituents of a gas stream; e.g, sulfur and nitrogen oxidesand mercury vapor, is described in U.S. Pat. No. 5,733,360. The reactoris designed to pulse generate a corona by applying high voltages pulsesfor up to 100 nanoseconds to a plurality of corona discharge electrodes.

WO 2006/046003 describes several methods for coating substratesinvolving use of a plasma, including use of low pressure pulsed plasmato introduce monomers or monomers in combination with free radicalinitiators to initiate polymerization on a suitable substrate. Anatmospheric pressure diffuse dielectric barrier discharge assembly isused into which an atomized liquid containing the monomers is introducedso that a coating material is formed from atomized drops of from 10 to100 μm. An atmospheric pressure glow discharge plasma generatingapparatus using radiofrequency energized electrodes is described in WO03/084682.

A plasma coating apparatus and method are described in WO 02/28548.Liquid or solid atomized coating forming materials are introduced into aplasma discharge at atmospheric pressure and are useful for organiccoatings such as polyacrylic acid or perfluoro compounds in addition tosilicon-containing monomers.

Corona effects are not always considered beneficial and may in factcause arcing, or the breakdown of the corona. In addition to thisbreakdown, the corona effect may be too strong to successfully onlysingly charge a complex molecule. When molecules ionize at a higherlevel, they may break apart and lose structural and functionalproperties.

One disadvantage of depositing materials generated in plasmas fromliquid solutions is that any solvent present is typically depositedalong with the intended material, creating unintended structures. Formost processes where corona discharge can occur during plasmageneration, efforts are usually taken to reduce the corona effect ratherthen using this effect as a deposition technique.

Deficiencies in the Art

The loss of functional and/or physical properties of plasma surfacedeposited organic molecules points to the need to develop methods ofmaintaining desirable biological activities of immobilized materials.Attempts to engineer biological coatings on a range of substrates, suchas plastics, metals, polymers, and ceramics have met with limitedsuccess and generally have failed to deposit biologically active agentson surfaces without compromising desired activity.

SUMMARY OF THE INVENTION

The present invention relates to a molecular plasma deposition methodfor non-destructively coating biological agents on substrate surfaces.The method employs a modified IPD apparatus utilizing electric fieldsand vacuum to lay down a biological coating on virtually any conductivesurface, and many non-conductive surfaces. A corona plasma moleculardischarge is generated from a highly charged conductive tip. The methodis applicable to deposition of a wide range of organic and inorganicmaterials, which are dispersed as solutions or suspensions from theconductive tip.

There are several features of the described method that differ fromconventional uses of either corona discharges or ionic plasmadepositions. The method employs solutions or suspensions of thematerials to be deposited. This allows a wide range of organic as wellas inorganic materials to be used, including elements and compounds. Theliquids are atomized through a small pointed orifice maintained at ahigh voltage so that an ionized plasma is dispensed from the orifice. Ina subsequent step of the method, the ionized plasma is directed into anevacuated chamber where an oppositely biased substrate is located,causing the material to be deposited where it becomes bonded to thesurface of the substrate.

An important feature of the method is the deposition of biologicallyactive agents onto a surface with little or no alteration of structureor functional characteristics. The method is equally applicable toinorganic materials, elements and select compositions, which are nototherwise amenable to coating processes. Due in part to the wide rangeof materials that can be deposited by this method, the ability to modifyor bio-engineer different surfaces is significantly expanded.

The known characteristics of the corona effect under atmosphericconditions and the advantages of ionic plasma deposition (IPD) methodsin coating processes have been used to develop a novel corona plasmadeposition process and coating method. An important aspect of theinvention is the ability to use a corona generated from a liquid orcolloidal composition to deposit a coating consisting of only thedesired component, without the solvent in which the material to bedeposited is dissolved or dispersed. Moreover, maintaining the originalstructural properties of a wide range of materials deposited from acorona generated molecular plasma was not expected, most notably asshown with a polypeptide enzyme, which maintained catalytic activityafter the molecular plasma deposition. Atomic bonds are not brokenduring the deposition process, a factor in retaining activity and/orstructural integrity of the deposited product.

In one embodiment, the process is carried out in part under atmosphericor partial pressure, and in part under vacuum. The deposition apparatusis designed to generate a corona from a solution or suspensionintroduced through a narrow electrified opening, such that a plasma isproduced in front of a small aperture that opens into a vacuum chamberhousing a substrate. Depending on the charge produced on the materialdispersed in the corona plasma, the substrate is wired as an oppositelycharged electrode on which the plasma particulates will deposit.

The basic structural characteristics of the deposited materials testedare not affected by the thickness of the deposit. This is in contrast tothe results obtained by Storey (Breakup of Biomolecules throughlow-energy ion Bombardment, Master's thesis, University of Missouri,Rolla, 1998) where more than 40 or so monolayers of glycine or argininedeposited by flooding a solution of the amino acids on gold caused lossof structure, as indicated by increasing difficulty in detecting thecarboxyl groups of the deposited amino acids as sample thicknessincreased. The disclosed molecular plasma method allows thickness to becontrolled from a mono-layer of desired material to micron thickness;i.e., 2-200 microns thick while maintaining structure and activity.

The apparatus for molecular plasma deposition can be modified toaccommodate a partial vacuum around the conductive tip where the coronais generated. This permits more efficient volatilization of the solventsuspending the dissolved or suspended material, so that only thematerial itself is drawn into the vacuum chamber housing to be depositedon the substrate and little if any solvent is present in the coating.This is necessary because, in biological applications, if the suspendingsolvent is also co-deposited, it may cause an adverse interaction withthe deposited material. For example, if a protein that one desires todeposit on a medical implant device is dissolved in methyl alcohol, anddeposited without volatilization of the alcohol before being placed inthe body, the residual alcohol may cause serious physiological problems.Where little or no solvent is drawn into the chamber, it is convenientto generate the corona under atmospheric pressure conditions.

As discussed, the new method is a molecular plasma procedure fordeposition of a biomolecule onto a substrate. A corona discharge plasmais generated under atmospheric or partial vacuum conditions from aliquid solution or suspension. The suspension is preferable colloidalsuspension for materials that have low solubility in organic or aqueoussolvents. Deposited materials may be an element, a compound, or any of anumber of biomolecules. The liquid solution or suspension is ejectedfrom a conductive point source at a high potential gradient and theresulting corona discharge is directed through an opening into anevacuated chamber where the ionized molecular plasma will be depositedonto a substrate which is maintained at an induced potential oppositefrom the relatively high potential at the point source where the coronais generated.

Generally, the conductive tip or point from which the colloidalsuspension is ejected provides a means for atomizing the solution orliquid suspension, so that there is ready formation of a coronadischarge at the high voltage tip. In many applications, one will preferto introduce the solution or liquid suspension from the tip underatmospheric conditions, but a low or partial vacuum can also be used,preferably 100 mTorr or higher. The charged plasma then passes through ahole or orifice into an evacuated chamber; e.g., at 40 mT or less,housing a substrate held at a voltage substantially opposite to thevoltage at the conductive tip. The ionized molecules in the coronaplasma then deposit onto the substrate in the chamber, which is at alower pressure than at the conductive tip where the corona is generated.

It is important to recognize that the substrate is under vacuum,typically less than 100 mTorr, preferably 40 mTorr or lower, and that ifthe plasma corona is formed at a tip also under reduced pressure, thesubstrate must be in a reduced pressure atmosphere such that the plasmacan be effectively drawn into the substrate housing and deposited ontothe substrate. The vacuum around the substrate is typically in the rangeof 40-0.1 mTorr. Additionally, the substrate must be oppositely biasedin order to effect deposition from the ionized molecular plasma, whichmay be positive or negative depending on the material in the colloidalsuspension. The ions formed in the discharge may be positive ornegative. This discharge will determine the bias of the substrate; e.g.,if a positive corona is used, the substrate must be negatively biased.

The amount of bias imposed on the substrate will depend on the substrateand on the area for deposition. In the examples provided herein, thesubstrates are approximately 4 cm². The bias of the substrate isconstant regardless of the size of the deposition; however, the largerthe area, the higher is the current necessary to maintain constantvoltage. Voltage applied to the substrate may range as high as 60 kV,which may be positively or negatively biased; i.e., opposite to thevoltage at the conductive tip where the corona is generated. Typicalvoltages range from +15 kV through −15 kV. Where the substrate isgrounded, the voltage will be zero at the substrate.

The method relies in part on efficiently generating an ionized plasma.This is accomplished by atomizing a liquid solution or suspension of thematerial desired for deposition through a sharp orifice or tip. This istypically a small diameter tube or needle that is imposed with a highvoltage. An exemplary high voltage on an 18 gauge metal needle with aninside diameter of approximately 0.83 mm, for example, may be about−5000 volts. In this example, the voltage applied to the substrate istypically in the range of about 5000 or less volts or is zero ifconnected to ground.

Yet another aspect of the invention is the ability to control the coronaeffect for more efficient processing; e.g., for engineering surfaces.The method takes advantage of the physics of the corona effect todeposit ionized material onto a substrate, so that the material isionically or covalently bonded to a substrate surface. This is a fast,easy way to produce an adherent coating and to structure a surface usinga non-destructive technique.

The described apparatus can be used to effectively deposit a wide rangeof materials without significantly altering the physical, functional orchemical characteristics, by generating material into a corona plasmafrom a liquid solution or suspension. Materials such as proteins arepreferably ionized from colloidal suspensions, due to their limitedsolubility in most solvents. Thymine, cytosine, adenine and guanine withrespective water solubilities of 4.5 g/L, insoluble, 0.5 g/L andinsoluble, are more efficiently deposited as aqueous suspensions. Almostany organic or inorganic material can be dissolved or suspended in somesolvent or mixture of solvents. Materials include proteins, amino acids,peptides, polypeptides, nucleic acids and nucleic acid bases such aspurines and pyrimidines, and the like. Examples of inorganic materialsinclude compounds such as metals and metal oxides, e.g., copper oxide,elements, including carbon. In general, biological and non-biologicalmaterials that can be prepared as a solution or liquid suspension willbe suitable materials for deposition. While colloidal suspensions may bepreferable for some biomolecules, microparticulate suspensions may alsobe suitable, depending on the material, thus creating ionized molecularplasmas from liquids containing micron- or larger sized particles.Examples of material that are preferably deposited from molecularplasmas generated from colloidal suspensions include, but are notlimited to: DNA, RNA, graphite, antibiotics, growth factors, growthinhibitors, viruses, inorganic compounds, catalysts, enzymes, organiccompounds, and elements. Catalase is one example of a polypeptide enzymethat can be deposited by this method without loss of catalytic activity.Other proteins expected to be deposited by this method without loss ofbiological activity include SNAP-tag fusion proteins, hAGT fusionproteins glucose binding protein, glutamine binding protein and lactatedehydrogenase. Oxidodreductases and oxidases such as glucose oxidase arealso expected to be deposited without loss of activity. Nucleotide basessuch as guanine, thymine, adenine, cytosine, and uracil are examples ofDNA and RNA bases that can be deposited onto a substrate from a coronagenerated plasma.

The solutions and liquid suspensions that are suitable for depositionmay be formulated from any of a number of aqueous or organic solvents,including pure water, alcohol and water/alcohol mixtures. Some materialsmay be prepared in less common solvents, such as DMSO or glycols. Forpreferred practice of the deposition procedure, one will select asolvent or combination of solvents in which the material can bedissolved or suspended, preferably as a colloidal suspension, and whichdoes not give rise to problems such as toxic or explosive fumes.

Definitions

Biomolecules are intended to include compounds and agents that have somebiological effect or use in the body and may include, withoutlimitation, proteins, peptides, amino acids, nucleic acids and compoundshaving drug activity or related to drug activity. As used herein,biomolecules also include carbon and carbon based compounds and elementsand compounds such as copper oxide that may be used as coating materialson medical devices.

Colloidal particles are finely divided particles approximately 10 to10,000 angstroms in size, dispersed within a continuous medium. Theparticles are not easily filtered and settle slowly over a period oftime.

Nano particles are about 100 nanometers or less in size.

Ionic Plasma Deposition (IPD) is the vacuum deposition of ionizedmaterial generated in a plasma, generally by applying high voltage orhigh current to a cathode target where the ionized plasma particles aredeposited on a substrate which acts as an anode.

A corona is produced by a process by which a current, perhaps sustained,develops from an electrode with a high potential in a neutral fluid,such as air, by ionizing the fluid to create a plasma around theelectrode. The ions generated eventually transfer a charge to nearbyareas of lower potential, or recombine to form neutral gas molecules.

Voltage bias is the potential, relative to earth ground, at whichsubstrate is held. Potential ranges from zero to 15,000 volts and can bepositive or negative. The potential on the substrate for biasing dependson the potential of the corona and is typically equal and opposite ofthis potential. Voltage may be as high as 60 kV but more typically is inthe range of 5-10 kV. Where the corona plasma is +5000 volts, biasvoltage for the substrate will be −5000 volts, all relative to earthground.

As used herein, substantial or substantially means that a range isintended, on the order of plus or minus ten percent and is not intendedto be limited to an exact number; for example, substantial function mayinclude different or less than original function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sketch of the molecular plasma deposition apparatus: vacuumchamber 1; high voltage power supply 2, substrate holder 3; substrate 4;high voltage power supply 5; needle 6; feeder tube to needle 7; orifice15 into reservoir 8; colloidal liquid suspension 9.

FIG. 2 is a sketch of a modification of the apparatus of FIG. 1: vacuumchamber 1; high voltage power supply 2; substrate holder 3; substrate 4;high voltage power supply 5; needle 6; feeder tube 7; orifice 15 intoreservoir 8; liquid suspension 9; secondary chamber 10; secondarychamber gas supply 11; secondary chamber gas supply line 12; pressureregulator 13; gas line from regulator 14.

FIG. 3 is a representation of the electric field equation for a pointcharge.

DETAILED DESCRIPTION

The present invention takes advantage of the corona effect and theeffect of corona discharge in creating a charged plasma that can bedirected to a substrate surface. The basic apparatus is shown in FIG. 1and FIG. 2. In an exemplary procedure, a high voltage of 5 kV or higheris applied to the needle or other hollow bore, sharp pointed, conductivematerial. A solution or liquid suspension is passed through the hollowbore. The high electric field at the tip of the needle causesatomization of the liquid as the result of the corona effect. Themolecules in the solution or suspension become charged, yet remainintact. The needle is positioned in front of a grounded, differentiallypumped high vacuum system with a small hole in the chamber housing thesubstrate. The substrate is placed inside the evacuated chamber at apotential opposite or nearly opposite to that imposed on the needle oris set to ground (zero). The charged molecules within the corona travelthrough the opening toward the substrate and become deposited orattached to the substrate, becoming ionically or covalently bonded.

The entire apparatus is enclosed in an environmentally controlledchamber into which selected gases such as oxygen or nitrogen may beintroduced; for example, if oxidation is desired, to control depositionrate, or to perform the deposition in an inert atmosphere. Mixtures ofgases may be introduced, including other inert gases such as xenon,argon, helium or combinations of gases.

The molecular plasma generation process can also be run at lower thanatmospheric pressures, i.e., under reduced pressure, in the presence ofgases other than atmospheric, (e.g., argon or oxygen backgroundatmosphere). When the molecular plasma at the conductive tip isgenerated under reduced pressure, the pressure in the chamber housingthe substrate must be lower so that the plasma discharge passes readilythrough the opening into the chamber housing the substrate, as shown inFIG. 1.

As shown in FIG. 1, the molecular plasma generation apparatus provides asystem for producing a plasma discharge under atmospheric conditions bypassing a liquid colloidal suspension 9 through a discharge needle 6 ata high voltage 5. The resulting atomized liquid forms an ionized plasmain the atmosphere. The plasma passes through an orifice 15 in the vacuumchamber 1 housing the substrate 4 on the substrate holder 3. A powersupply 2 provides voltage to the substrate 4 at a voltage opposite tothat provided by the power supply 5 to the discharge needle 6.

FIG. 2 illustrates an alternative embodiment of a system for producingan ionized plasma discharge. A reservoir 8, feeds a solution or liquidsuspension of the material 9 through an orifice 15 for deposition of thecolloidal material on the substrate 4. The liquid is passed through thehighly charged needle 6 from the power supply 5. In this embodiment, thefeeder and needle are housed in a second chamber 10 which can bepressure regulated by a pressure control 13 through the opening 14 intothe secondary chamber. The atmosphere within the secondary chamber 10can be modified from a gas container 11 having a conduit 12 passingthrough the regulator 13. The vacuum chamber 1 is maintained at a lowerpressure than in chamber 10. The substrate 4 is biased using the powersupply 2 at a voltage opposite to that supplied by power supply 5 to theneedle 6.

Liquid suspensions or solutions may be prepared in organic or inorganicliquids, which should not be toxic or flammable. Most materials arepreferably prepared as aqueous solutions or may be prepared in organicacids such as acetic acid, propionic acid, halogen substituted aceticacid, oxalic acid, malonic acid and/or hydroxycarboxylic acids alone orwith water. Liquid mixtures may include salts or organic/water misciblepreparations. Examples of alcohols include ethanol, methanol, andketones such as acetone, DMF, THF and methylethylketone. Amino acids,for example, may be water soluble at low concentrations but formcolloidal suspensions at higher concentrations. Lysine and threonine arehighly water soluble while tyrosine has a limited solubility of about0.045 g/100 ml at 25° C.

BACKGROUND DESCRIPTION OF CORONAS AND ELECTRICAL DISCHARGES

Corona discharge of both the positive and negative variety is commonlycharacterized as ionization of a neutral atom or molecule in a region ofstrong electrical field typically in the high potential gradient near acurved electrode, creating a positive ion and a free electron. Theelectric field then separates and accelerates the charged particlespreventing recombination and imparting each particle with kineticenergy. Energized electrons, which have a much higher charge/mass ratioand so are accelerated to a higher velocity, may create additionalelectron/positive-ion pairs by collision with neutral atoms. These thenundergo the same separating process, giving rise to an electronavalanche. Both positive and negative coronas rely on electronavalanches. FIG. 3 illustrates a typical point charge formed in a strongelectrical field.

The energy of these plasma processes is converted into initial electrondissociations to seed further avalanches. An ion species created in thisseries of avalanches, which differs between positive and negativecoronas, is attracted to an uncurved electrode, e.g., a flat surface,completing the circuit, and sustaining the current flow.

A corona is a process by which a current, whether or not sustained,develops from an electrode with a high potential gradient in a neutralfluid, usually air. When the potential gradient is large enough at apoint in the fluid, the fluid at that point ionizes and it becomesconductive. If a charged object has a sharp point, the air around thatpoint will be at a higher gradient than elsewhere, and can becomeconductive while other points in the air do not. When the air becomesconductive, it effectively increases the size of the conductor. If thenew conductive region is less sharp, the ionization may not extend pastthis local region. Outside of this region of ionization andconductivity, the charged particles slowly find their way to anoppositely charged object and are neutralized. On the other hand, if thegeometry and gradient are such that the ionized region continues to growinstead of stopping at a certain radius, a completely conductive path isformed, and a momentary or continuous spark or arc occurs.

Corona discharge usually involves two asymmetric electrodes, one highlycurved, such as the tip of a needle, or a narrow wire, and one of lowcurvature, such as a plate, or the ground. The high curvature ensures ahigh potential gradient around one electrode inn order to effectivelygenerate a plasma.

Coronas may be positive or negative. This is determined by the polarityof the voltage on the highly-curved electrode. If the curved electrodeis positive with respect to the flat electrode the corona is positive;if the electrode is negative, a negative corona exists. The physics ofpositive and negative coronas are strikingly different. This asymmetryis a result of the large difference in mass between electrons andpositively charged ions, with only the electron having the ability toundergo a significant degree of ionizing inelastic collisions atstandard temperatures and pressures.

A negative corona is manifested as a non-uniform corona, varyingaccording to the surface features and irregularities of the curvedconductor. It often appears as tufts of corona at sharp edges, thenumber of tufts changing with the strength of the field. The form ofnegative coronas is a result of its source of secondary avalancheelectrons. It appears a little larger than the corresponding positivecorona, as electrons are allowed to drift out of the ionizing region,allowing the plasma to continue some distance beyond it. The totalnumber of electrons and electron density is much greater than in thecorresponding positive corona; however, the electrons are at apredominantly lower energy, owing to being in a region of lowerpotential-gradient. Therefore, while for many reactions the increasedelectron density will increase the reaction rate, the lower energy ofthe electrons means that reactions which require a higher electronenergy may take place at a lower rate.

A positive corona is manifests as a uniform plasma across the length ofa conductor. It is often observed as a blue/white glow, although much ofthe emission is in the ultraviolet. The uniformity of the plasma is dueto the homogeneous source of secondary avalanche electrons. With thesame geometry and voltages, a positive corona appears somewhat smallerthan the corresponding negative corona, owing to the lack of anon-ionizing plasma region between the inner and outer regions. Thereare many fewer free electrons in a positive corona, perhaps a thousandthof the electron density, and a hundredth of the total number ofelectrons, compared to a negative corona, with the exception of the areaclose to the curved electrode where electrons are highly concentrated.This region has a high potential gradient, causing the electrons to havehigher energy. Most of the electrons in a negative corona are in outer,lower energy field areas.

In a positive corona, secondary electrons, giving rise to additionalavalanches, are generated predominantly in the fluid itself, in theregion outside the plasma or avalanche region. They are created byionization caused by the photons emitted from that plasma in the variousde-excitation processes occurring within the plasma after electroncollisions. The thermal energy liberated in those collisions createsphotons which are radiated into the gas. The electrons resulting fromthe ionization of a neutral gas molecule are then electrically attractedback toward the curved electrode and into the plasma, cycling theprocess of creating further avalanches inside the plasma.

The positive corona is divided into two regions, concentric around thesharp electrode. The inner region contains ionizing electrons, andpositive ions, acting as a plasma, the electrons avalanche in thisregion, creating many further ion/electron pairs. The outer regionconsists almost entirely of the slowly migrating massive positive ions,moving toward the uncurved electrode along with, close to the interfaceof this region, secondary electrons, liberated by photons leaving theplasma, being re-accelerated into the plasma. The inner region is knownas the plasma region, the outer as the unipolar region.

A negative corona is manifested as a non-uniform corona, varyingaccording to the surface features and irregularities of the curvedconductor. It often appears as tufts of corona at sharp edges, thenumber of tufts altering with the strength of the field. The form ofnegative coronas is a result of its source of secondary avalancheelectrons. The negative corona appears a little larger than thecorresponding positive corona, due to drifting of the electrons from theionizing region, so that the plasma continues some distance beyond it.The total number of electrons, and accordingly the electron density, ismuch greater than in the corresponding positive corona. The electronsare lower energy that those in a positive corona because they are in aregion of lower potential-gradient.

Negative coronas are more complex than positive coronas in construction.As with positive coronas, the establishing of a corona begins with anexogenous ionization event generating a primary electron, followed by anelectron avalanche.

The difference between positive and negative coronas is in thegeneration of secondary electron avalanches. In a positive corona theavalanches are generated by the gas surrounding the plasma region, thenew secondary electrons traveling inward, while in a negative coronathey are generated by the curved electrode itself, the new secondaryelectrons traveling outward.

An additional structural feature of negative coronas is the outwarddrift of the electrons, where they encounter neutral molecules and maycombine with electronegative molecules such as oxygen and or water vaporto produce negative ions. These negative ions are then attracted to apositive uncurved electrode, completing the ‘circuit’.

A negative corona can be divided into three radial areas, around thesharp electrode. In the inner area, high-energy electrons inelasticallycollide with neutral atoms and cause avalanches, while outer electrons,usually of a lower energy, combine with neutral atoms to producenegative ions. In the intermediate region, electrons combine to formnegative ions, but typically have insufficient energy to cause avalancheionization. They remain part of a plasma owing to the differentpolarities of the species present, and the ability to participate incharacteristic plasma reactions. In the outer region, only a flow ofnegative ions and, to a lesser and radially-decreasing extent, freeelectron movement toward the positive electrode takes place. The innertwo regions are known as the corona plasma. The inner region is anionizing plasma, the middle a non-ionizing plasma. The outer region isknown as the unipolar region.

As discussed, the corona principal has been used to create anapproximately infinite electric field at the point of a sharp needle.For practical purposes, it can be assumed that the tip of the device isatomically sharp and closely approximates a point charge. This isbecause as r goes to zero, E approaches infinity. A corona effect isinitiated at the tip of the device.

The energy of the electrons and relation to the distance from the pointsource of generation is based on the electric field of a point chargederived from Coulomb's law. This law states the electric field from anynumber of point charges can be obtained from a vector sum of theindividual fields. A positive number is taken to be an outward field;the field of a negative charge is toward it. This can be shown inequation 1 and illustrated in FIG. 3:

$\begin{matrix}{E = {\frac{F}{q} = {\frac{k\; Q_{source}\mspace{14mu} q}{{qr}^{2}} = \frac{{kQ}_{source}}{r^{2}}}}} & 1\end{matrix}$

EXAMPLES

The following examples are intended only as illustrations of theinvention and are in no way to be considered limiting for what isdescribed and taught herein.

Example 1—Apparatus for Molecular Plasma Deposition

An exemplary apparatus includes a vacuum chamber with a small aperture,and a small bore, metallic needle connected to a tube connected to areservoir holding a liquid suspension or solution of the materialdesired to be deposited. The reservoir is at atmospheric pressure. Apower supply with the ability to supply up to 60 kV can be employed;however, as used in the examples herein, the voltage attached to theneedle is typically −5000 volts to +5000 volts. A substrate inside thevacuum chamber, is centered on the aperture with a bias from −60 kVthrough −60 kV, including ground. The apparatus is illustrated in FIG.1.

Example 2—Apparatus for Molecular Plasma Generation under SelectedEnvironments

The apparatus illustrated in FIG. 2 can be modified such that theneedle, tube, and reservoir are disposed in an enclosure that excludesair, but allows for the controlled introduction of other gases.Optionally selected gases include argon, oxygen, nitrogen, xenon,hydrogen, krypton, radon, chlorine, helium, ammonia, fluorine andcombinations of these gases.

Example 3—Apparatus for Corona Discharge Generation Under ReducedPressure

In the apparatus shown in FIG. 1, the pressure differential between thecorona discharge and the substrate is about one atmosphere. The outsidepressure of the vacuum chamber is approximately 760 Torr, whereaspressure in the area of the substrate is approximately 0.1 Torr.

The apparatus shown in FIG. 2, on the other hand, can be optionallyoperated at a pre-determined pressure above or below atmosphericpressure. While atmospheric pressure is generally preferred forgeneration of the plasma, reduced pressure up to about 100 mTorr may insome instances provide satisfactory depositions.

Example 4—Molecular Plasma Deposition of Amino Acids

This example illustrates deposition of a suspension of amino acids ontoa gold rod. A colloidal suspension of a mixture of the amino acidsglycine (solubility of 20 g/l at 25° C.), alanine (166.5 g/l), valine(88.5 g/l), leucine (24.26 g/l) and arginine (235.8 g/l) in water wasdeposited using the apparatus of Example 1 onto a gold covered rod, ⅛″in diameter and approximately 0.75 cm². The power supply was attached toa 304 stainless steel 18 gauge needle and set at −5000 V. The goldsubstrate was set at a potential of 5000 V. The substrate was centeredon the hole in the chamber and placed 5 cm from the hole. The vacuumchamber was pumped to 40 mTorr and the flow of the colloidal suspensionwas initiated. The deposition was carried out for 30 min.

The coated rod was placed in a time-of-flight secondary ion massspectrometer (TOF-SIMS) and the components were analyzed forcomposition. Results showed that the amino acids were deposited intactand ionically bonded to the substrate. Mass over charge calculations inconjunction with the time of flight spectrometry were used to calculatethe masses of the incoming species. These calculations were used tointerpret the spectra from the SIMS. The m/q data showed the amino acidsbeing ejected intact from the surface.

In a control comparison experiment, the substrate was dipped into theamino acid mixture and analyzed by TOF-SIMS as above. These spectra weresubtracted from amino acid spectra generated from corona deposition inorder to isolate any effects that occurred due only to the depositionmethod. Fragmentation was observed in both spectra, and aftersubtraction, it was determined that the fragmentation was an effect ofthe analytic technique, not the deposition technique because thefragmentation occurred equally in both spectra.

Example 5—Molecular Plasma Deposition of Graphite

A colloidal suspension of graphite powder in isopropyl alcohol (10g/1001 ml) was deposited onto an aluminum oxide substrate using theapparatus shown in FIG. 1.

The power supply was attached to a 304 stainless steel 18 gauge needleand set at −5000V. The aluminum oxide substrate was connected to ground.The substrate was centered on the hole in the chamber and placed 5 cmfrom the hole. The vacuum chamber was pumped to 40 mTorr and the flow ofthe colloidal suspension was initiated. The deposition was carried outfor 30 minutes. The substrate was removed from the chamber and a simpleohm meter resistance test performed. Resistance of the substrate changedfrom infinite to 1 ohm over the 30 min deposition period.

Example 6—Molecular Plasma Deposition of Copper Oxide

A colloidal suspension of copper oxide powder in water (10 g/100 ml) wasprepared. Using the apparatus illustrated in FIG. 2, the high voltagepower supply was attached to a 304 stainless steel, 18 gauge needle setat −10,000V. The substrate was 304 stainless steel and set at apotential of 5000 V. The substrate was centered and placed 5 cm from thehole in the chamber. The chamber was pumped to 40 mTorr and the flow ofthe colloidal suspension initiated. The deposition onto the substratewas allowed to proceed for 10 minutes. At the end of the depositionprocess, the substrate was removed from the chamber and a simple tapetest showed good adhesion of the deposited copper oxide. Good adhesionbetween the substrate and the copper oxide were confirmed by repeatingthe tape test and by observing that after sonicating the coated samplefor 10 min there was no evidence of flaking or sloughing.

Example 7—Molecular Plasma Deposition of RNA and DNA Bases

A colloidal suspension of guanine, adenine, cytosine, uracil and thyminein water (each at 5 g/100 ml) was deposited onto gold covered rod havinga surface of approximately 0.75 cm² area, ⅛″ diameter, using theapparatus of Example 1. The power supply was attached to a 304 stainlesssteel 18 gauge needle and set at −5000V. The gold substrate was set at apotential of 5000 V. The substrate was centered on the hole in thechamber and placed 5 cm from the hole. The vacuum chamber was pumped to40 mTorr and the flow of the colloidal suspension was initiated. Thedeposition was carried out for 30 min.

The coated rod was placed in a time-of-flight secondary ion massspectrometer (TOF-SIMS) and the components were analyzed forcomposition. Results showed that the DNA bases were deposited intact andionically bonded to the substrate. Mass over charge calculations inconjunction with the time of flight spectrometry were used to calculatethe masses of the incoming species.

These calculations were used to interpret the spectra from the SIMS. Them/q data showed the bases being ejected from the surface as beingintact. Spectra from another deposition method (dipping the substrate ina mixture containing the bases) ware also analyzed as a control to thebases deposited using the corona effect. The spectra was subtracted fromthe corona effect spectrum to isolate any effects that occurred due onlyto the deposition method. Fragmentation was observed in both spectra,and once subtracted, it was determined this observation was a product ofthe analytic technique and not the deposition technique because thefragmentation occurred equally in both spectra.

Example 8—Molecular Plasma Deposition of Catalase

25 ml of a 2× crystallized bovine liver catalase (Sigma C100-58MG;056K7010) colloidal suspension in water with 0.1% thymol was prepared.Protein concentration was 33 mg/ml with an activity of 4.1×10⁴ U/ml.

Using the apparatus illustrated in FIG. 2, the high voltage power supplywas attached to a 304 stainless steel, 18 gauge needle set at −5000V.The substrate was an aluminum oxide disk ¼″ thick by 1.5″ in diameter,having an area of approximately 11 sq cm and set at a potential of 5000V. The substrate was centered and placed 5 cm from the hole in thechamber. The chamber was pumped to 40 mTorr and the flow of thecolloidal suspension initiated. The deposition onto the substrate wasallowed to proceed for 10 minutes. At the end of the deposition process,the substrate was removed from the chamber and the sample was placed ina 5% solution of hydrogen peroxide. The results showed the catalysis ofthe hydrogen peroxide by the catalase, producing bubbling of oxygen fromthe surface, showing that the enzyme remained intact throughout thedeposition process.

The deposition was repeated twice under the same conditions, except thatafter the substrate was removed from the chamber, the samples wereplaced in an ultrasonic water bath for 10 min. Additionally, one of thesamples was maintained at 10° C. for 72 hr after removal from the bath.In each case, exposure of the sample to a 5% solution of hydrogenperoxide produced bubbling of oxygen from the surface of the substrate.The ultrasonic treatment did not affect the deposited material,indicating that a stable, adherent coating of catalase had beendeposited.

1. A molecular plasma method for deposition of a biomolecule onto asubstrate, comprising, generating an ionized molecular plasma coronaunder atmospheric conditions from a biomolecule in a liquid solution orsuspension ejected from a conductive point connected to a voltage sourcesufficient to generate the ionized molecular plasma wherein thebiomolecule in solution or suspension becomes charged, yet remainsintact; and directing the generated plasma through an orifice of anevacuated chamber housing a substrate at an induced potential oppositeto the conductive point; wherein the biomolecule is deposited onto thesubstrate without significantly altering the physical, functional orchemical characteristics.
 2. The method of claim 1 wherein thesuspension is colloidal.
 3. The method of claim 1 wherein the liquid iswater, alcohol or mixtures thereof.
 4. The method of claim 1 wherein thegenerated corona discharge plasma comprises a positive or negativelycharged plasma.
 5. The method of claim 1 wherein the biomolecule is anamino acid, a nucleic acid base, or a polypeptide.
 6. The method ofclaim 1 wherein the biomolecule is selected from the group consisting ofcarbon, copper oxide, an amino acid, an RNA or DNA base, catalase andmixtures thereof.
 7. The method of claim 1 wherein the biomolecule is atleast one of glycine, alanine, valine, leucine, arginine or mixturesthereof.
 8. The method of claim 1 wherein the biomolecule is at leastone of guanine, adenine, thymine, cytosine, uracil or mixtures thereof.9. The method of claim 2 wherein the colloidal suspension is comprisedof particles having a size range between about 100 angstroms and about10,000 angstroms.
 10. The method of claim 1 wherein the substrate is ametal, ceramic or a plastic.
 11. A molecular plasma method fordeposition of a biomolecule onto a substrate, comprising, atomizing asolution or liquid suspension of a biomolecule into a partial vacuumfrom a conductive tip at a voltage sufficient to generate a plasmacontaining the intact charged biomolecule; and directing the plasmathrough an orifice into an evacuated chamber housing a substrateconnected to ground or held at a voltage opposite to the voltage of theconductive tip; wherein the biomolecule deposits onto the substrate. 12.The method of claim 11 wherein the biomolecule is selected from thegroup consisting of carbon, copper oxide, an amino acid, an RNA or DNAbase, catalase and mixtures thereof.
 13. The method of claim 11 whereinthe biomolecule is at least one of glycine, alanine, valine, leucine,arginine or mixtures thereof.
 14. The method of claim 11 wherein thebiomolecule is at least one of guanine, adenine, thymine, cytosine,uracil or mixtures thereof.
 15. The method of claim 11 wherein theliquid suspension is a colloidal suspension comprised of particleshaving a size range between about 100 angstroms and about 10,000angstroms.
 16. The method of claim 11 wherein the solution or liquidsuspension comprises water, alcohol, glycol or combinations thereof. 17.The method of claim 11 wherein the vacuum in the chamber is less than100 mTorr.
 18. The method of claim 11 wherein the voltage applied to theconductive tip is positive or negative in the range of 15,000 volts. 19.The method of claim 11 wherein the liquid is atomized into a vacuum inthe range of 100 mTorr up to atmospheric pressure.
 20. The method ofclaim 11 wherein the biomolecule is deposited on the substrate withoutsignificantly altering the physical, functional or chemicalcharacteristics.